review on validation of liquid chromatography mass...

1

Review on validation of liquid chromatography–mass spectrometry methods

... and more!

Ivo Leito

University of Tartu

[email protected]

Euro Mass Spectrometry 2017

sisu.ut.ee/lcms_method_validation/

LC-MS as technique

• The most powerful technique for determination of

low levels of organics in difficult matrices

• LC-MS: many adjustable parameters

– In LC

– In MS

Euro Mass Spectrometry 2017 2

Checking that the method

performs as required is not trivial!

Validation is BIG in LC-MS!


Guidelines?

Validation guidelines

• Generally very useful, but ...

– Often inconsistent terminology

• e.g. accuracy vs trueness, ...

– Often very general

• Choosing concentrations? How many replicates? Spiking

levels? How many days? ...

– Sometimes conflicting recommendations

– Usually LC-MS is not addressed

• Except e.g. 2002/657/EC

Euro Mass Spectrometry 2017 4 sisu.ut.ee/lcms_method_validation/

Our aims were:

1. Give a critical overview of the state of the art of

LC-MS method validation

– Including comparisons of the relevant guidance

materials

2. Draw attention to the LC-MS specific aspects

– e.g., ionization suppression by matrix effects

3. Give recommendations on problematic issues in

validation of LC-MS methods


Outcome:

Two-part tutorial

review in

Anal. Chim. Acta


- Tutorial review on validation of liquid chromatography–mass spectrometry

methods: Part I. A. Kruve, R. Rebane, K. Kipper, M.-L. Oldekop, H. Evard, K.

Herodes, P. Ravio, I. Leito. Anal. Chim. Acta 2015, 870, 29-44.

http://dx.doi.org/10.1016/j.aca.2015.02.017

- Tutorial review on validation of liquid chromatography–mass spectrometry

methods: Part II. A. Kruve, R. Rebane, K. Kipper, M.-L. Oldekop, H. Evard, K.

Herodes, P. Ravio, I. Leito. Anal. Chim. Acta 2015, 870, 8-28.


Features

• Specific LC-MS issues

– Ionization, matrix effects, MSn selectivity, ...

• Main validation guidelines are reviewed and compared

– With every performance parameter

• Recommendations are given how to determine

performance parameters

– Synthesis from guidelines and our experience

• General workflow of LC-MS method validation is

presented



Is (100% - ST%) of 2/3 of the samples lower then 1/5 of the expected relative

standard uncertainty?

Analyse at least 20 representative blank samples. Analyse representative

blank samples fortified with substances which are likely to interfere with the

quantification and/or identification of the analyte.

Any false identification? Analyte identification hindered by interferent?

Notable influence on quantitation?

Analyse CRM-s or spiked samples with known concentration.

Calculate bias and compare to legal requirements if available.

Analyse matrix containing solutions at 10 analyte concentrations in duplicates

a) Visual assessment b) analysis of residuals c) LoF test if neccessary.

Analyse independently prepared subsamples at least at 3 concentration

levels over the linear range (5-10 replicates each). Repeatability

measurements are done on the same day, intermediate precision

measurements over several months.

Calculate relative standard deviation of repeatability and intermediateprecision and test it for „fit-for-

purpose“.

6 calibration points in the LoD range should be used. Measurements should be done on 5 different days over a long

time period.LoD = 3.3∙σ/S, LoQ = 10∙σ/S.

The most conservative result should be used and compared to the legal

requirements if available.

Select method parameters having thestrongest influence on your method

performance. Test the method performance after small changes in

selected parameters.

In the method performance still acceptable?

Estimate method conditions and test analyte and IS stability. At least 3

replicates at two concentration levels should be evaluated under stress

conditions

StartMethod

optimization

Ok!

Ok!

Ok!

Ok!

Ok!

Ok!

Not Ok!

Not Ok!

Ok!

Final Reporting

Method issuitable for

routine application

Not Ok!

Not Ok!

Not Ok!

Not Ok!

Not Ok!

Stability

Selectivity

Linearity

Precision

Trueness

LoD, LoQ

Robustness

Tutorial review on estimating LoD on the example

of LC/MS


- Tutorial on Estimating Limit of Detection on the example of LC-MS analysis:

Part I. H. Evard, A. Kruve, I. Leito. Anal. Chim. Acta 2016, 942, 23-39.


- Tutorial on Estimating Limit of Detection on the example of LC-MS analysis:

Part II. H. Evard, A. Kruve, I. Leito. Anal. Chim. Acta 2016, 942, 40-49.



On-line course: LC/MS Method Validation


Online LC/MS Validation course

• Validation: General

• Performance parameters

– Selectivity, Identity

• Incl specifics of LC/MS

– Linearity, linear range, sensitivity

– Precision, Trueness, Accuracy

• Including matrix effects

– Stability

– LoD, LoQ, CCa, CCb

• Including specifics of LC/MS

– Ruggedness, Robustness

• Glossary

• Literature


Course contents • Theoretical basis as well as practical skills

• Detailed and example-based treatment

• More than 50 short video lectures by 6 teachers

– Excel videos demonstrating calculations

• Numerous tests and calculation exercises

– Understanding of main concepts

– Calculation exercises from real life situations

– Feedback is given

• On-line software (some functionality)


Videos ...

• Using slides:

... or ...

• Pen and whiteboard:


Discussion dialogues

• Two proffessionals discuss a problematic

situations: one asks for help and the other one

gives suggestions


Lab

videos


Excel videos for calculations

• Initial calculation files can be downloaded


Worked examples


Self-tests


Online validation software (some functionality is available)


„Usage modes“


LC/MS Method

Validation sisu.ut.ee/lcms_method_validation/

On-line course for independent

learning available any time

anywhere

On-line reference point

of terminology, explanations and

self-testing possibilities

Running as MOOC

with registered participation, on-line counseling, graded tests and certificate

Course material for running training courses e.g. by labs

Support to university teaching

Students can be directed here e.g. for

self-testing

MOOC: Autumn 2016 edition 303 registered participants from 61 countries

22 Euro Mass Spectrometry 2017

Overall completion rate: 55%

Completion rate of those who started: 75%

Interested?

• Without registration: the materials are freely

available 24/7

• Registered participation:

– Forums

– Graded tests

– Certificate



LC/MS Validation MOOC – second edition: Winter 2017-2018

Registration will open in autumn 2017

Many thanks to the team!

Course development:

Anneli Koit Maarja-Liisa Riin Karin Hanno Evard

Kruve Herodes Oldekop Rebane Kipper

Video, web design, admin:

Irja Helm Triin Marandi Lehti Pilt Esta Pilt Toomas Petersell

Juho Jalviste



Thank you for your attention!


Questions, requests:

[email protected]

LC/MS Validation MOOC – second edition: Winter 2017-2018

Registration will open in autumn 2017

review on validation of liquid chromatography mass...

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